Closed system respirometry may underestimate tissue gas exchange and bias the respiratory exchange ratio (RER).

Closed respirometry is a commonly used method to measure gas exchange in animals due to its apparent simplicity. Typically, the rates of O2 uptake and CO2 excretion (VO2 and VCO2, respectively) are assumed to be in steady state, such that the measured rates of gas exchange equal those at tissue level. In other words, the respiratory gas exchange ratio (RER) is assumed to equal the respiratory quotient (RQ). However, because the gas concentrations change progressively during closure, the animal inspires air with a progressively increasing CO2 concentration and decreasing O2 concentration. These changes will eventually affect gas exchange causing the O2 and CO2 stores within the animal to change. Because of the higher solubility/capacitance of CO2 in the tissues of the body, VCO2 will be more affected than VO2, and we hypothesize therefore that RER will become progressively underestimated as closure time is prolonged. This hypothesis was addressed by a combination of experimental studies involving closed respirometry on ball pythons (Python regius) as well as mathematical models of gas exchange. We show that increased closed duration of the respirometer reduces RER by up to 13%, and these findings may explain previous reports of RER values being below 0.7. Our model reveals that the maximally possible reduction in RER is determined by the storage capacity of the body for CO2 (product of size and specific capacitance) relative to the respirometer storage capacity. Furthermore, modeling also shows that pronounced ventilatory and circulatory response to hypercapnia can alleviate the reduction in RER.